Nucleic acid hybridization tests, also referred to as DNA or RNA probe tests provide new means of analyzing genetic information contained in test samples. Nucleic acid hybridization assays can thus provide important new clinical diagnostic capability and yield information on the genetic basis and susceptibility to disease. For example, hybridization assays have established relationships between viral infections and cancer. Prenatal diagnosis of genetic disease and detection of inherited disease traits have also been reported. Nucleic acid hybridization assays have also found application in the identification of slow growing and resistant infectious organisms [J. Skylar, Human Pathology, 16 (7), 654 (1985)].
Mixed-phase hybridization systems have typically been used to perform these assays. In mixed phase assays the hybridizations are performed on a solid phase such as nylon or nitrocellulose membranes. These assays can be cumbersome involving complicated, multi-step procedures. For example, the assays usually involve loading a membrane with sample, denaturing the DNA to create single-stranded molecules, fixing the DNA or RNA to the membrane, and saturating the remaining membrane attachment sites with heterologous nucleic acids to prevent the probe reagent from adhering to the membrane in a nonspecific manner. All of these steps must be done before performing the actual hybridization. The conventional membrane based test procedures are time consuming (4-12 hours) and complex--requiring multiple reagent additions, wash procedures, and hands-on manipulations [see, for example, M. J. Gore, Clin. Chem. News, 12 (6), 1 (1986)].
Membrane-based hybridizations are not always directly useful for crude samples. The membranes are subject to clogging. Moreover, crude samples contain proteins, lipids, mucopolysaccharides, etc., which compete for binding sites on the membranes and, when fixed, reduce the binding capacity of the membrane and contribute to nonspecific binding of reporter reagents. These competing interactions cause unacceptable background and diminished test response. Furthermore, target DNA is found typically in minute quantities (&lt;10.sup.-15 M) in most test samples, since only a few copies of target DNA are present in each cell. For these reasons, for clinical diagnostic applications, the nucleic acids in test samples must be at least partially purified and concentrated prior to testing.
To circumvent these drawbacks, several new hybridization techniques have been reported in the literature. A. R. Dunn et al. [Cell, 12, 23-36 (1977)] along with M. Ranki et al. [Gene. 21, 77-85 (1983)] describe the application of sandwich hybridization procedures for probe tests. Sandwich hybridization assays generally involve the use of two probe reagents. One probe is attached to a solid phase support such as a membrane and the other probe is attached to a label. Both probes are complementary to a portion of the target nucleic acid. When target nucleic acid is present, the labeled probe will be bound to the solid phase support through the target nucleic acid and can then be detected after washing the solid phase support. M. Ranki et al., Current Topics in Microbiology and Immunology, 104, 307-318 (1983), report that the sandwich hybridization assays can be performed on crude samples. The sandwich hybridization technology can use various solid supports and has provided a way to reduce sample pretreatment complexity and to reduce the number of assay steps.
Despite gains realized in enhanced procedural simplicity and reduced sample pretreatment, sandwich hybridization assays continue to suffer from long equilibration times. This is primarily caused by the concentration dependence of hybridization reactions which dictates that longer equilibration times are required at lower target DNA and probe reagent concentrations (B. D. Hames and D. J. Higgins, eds., Nucleic Acid Hybridizations, pp 48-53, IRL Press, 1985).
Sandwich hybridization assays depend on two independent hybridization events. The reaction times are influenced by both the reporter probe and capture probe concentrations. Furthermore, the reaction rates are known to be slower on solid phase reagents than in solution. In addition, the equilibration procedures and wash steps, required in the assay to free the solid phase supports from unhybridized probe, also extend testing time and thus diminish clinical usefulness of the technology. Currently, the long assay times prevent test results from being used in cases of emergency or performed in time to be used during a patient's office appointment.
The most severe drawback of current hybridization assays for both diagnostic and research applications, has been inadequate test sensitivity. Generally, only a few copies of target nucleic acid are present in test materials of clinical interest. For example, infectious disease specimens generally contain only a few infectious organisms. The presence of from 1-1.times.10.sup.6 organisms is generally considered clinically important. Since each organism contains only a few copies of target nucleic acids per cell (4 to 100), the total target nucleic acid available ranges from approximately 10.sup.-16 to 10.sup.-22 moles. This is below the current limit of detection for hybridization assays. For this reason, hybridization assays have not been generally applicable to direct specimen testing but have found use in testing specimens in which the number of organisms has been increased by culturing.
Several methods which have recently been disclosed attempt to circumvent the constraint of limited copies of target nucleic acids. In a patent application, Ser. No. WO 84/02721, Kohne described the use of probes directed against RNA targets. Since many thousands of copies of target RNA can be produced in vivo during expression of a single copy of DNA, RNA probe tests would appear to be inherently more useful and thus applicable to direct specimen testing. However, RNA targets are particularly labile and subject to enzymatic digestion by the large quantities of ribonucleases found in samples.
K. B. Mullis and K. B. Mullis et al. U.S. Pat. No(s). 4,683,202 and 4,683,195, both issued July 28, 1987, respectively, disclose a gene amplification technique which can be used to multiply the number of DNA copies present in test samples. In this approach, the target DNA is replicated multiple times in vitro using a DNA polymerase enzyme. This way, the number of copies of DNA can be greatly increased (about 10.sup.6 -fold). Once amplified, the target DNA can then be tested using conventional hybridization assays. This technique, known as a polymerase chain reaction procedure, involves multiple steps which add time additional user manipulations and reagent costs to the overall hybridization assay.
Attempts to modify the original sandwich hybridization assay principle also have not afforded the required sensitivity. For example, in European Patent Application 154,505, J. E. Monahan et al. describe a sandwich hybridization format specifically designed to detect a specific single nucleotide change in the nucleotide sequence such as would occur as a result of a genetic mutation. During analysis, the target DNA is contacted with restriction endonucleases specifically selected to cleave or, alternatively, to conserve the integrity of the strand at the point of sequence change. Since the integrity of the target chain depends on the presence or absence of the nucleotide sequence change site, the integrity of the target chain is detected by determining whether the reporter probe remains attached to the solid phase immobilized capture probe during hybridization or is present in the solution following separation of the two phases. Alternatively, if detection depends on agglutination, settling rates or changes in agglutination could be used to signal the integrity of the target chain after treatment with restriction enzymes.
In European Patent Application 130,515 A2, Dattagupta et al, describe a dual hybridization assay concept for detecting nucleotide sequences in DNA samples. This approach is similar to that of Monahan et al. in that both make use of first and second nucleic acid probes, one of which is attached to a solid phase support complementary to adjacent sequences at a specific restriction site which is unique to the target DNA. The method is ideally suited to the detection of inherited diseases such as sickle cell anemia or to detect point mutations occurring in known gene sequences. However, both approaches suffer from inherent drawbacks. Both of these methods are limited in scope of application since probe reagents must be prepared complementary to sequences adjacent to a restriction site. Sequences distant from the restriction site cannot be used since the same enzyme can produce multiple cuts in large pieces of DNA. Applications of these assays are further limited in scope to gene sequences for which restriction enzymes are available. Furthermore, these assays are not suited for quantitative analysis. The absence of target DNA would appear as if restriction sensitive target were present since the partitioning of the solid phase probe would result in solubilized labeled probe.
While the techniques of Monahan et al, and Dattagupta et al. did achieve increased specificity, they did not achieve the desired increase in sensitivity. Furthermore, they do not suggest ways for enhancing detection of these labels. Neither the method of Monahan et al. nor of Dattagupta et al. extends the detection limits of sandwich hybridization assays.
Patent Application WO 86/03782 describes a sandwich hybridization assay having high sensitivity. One of the reagents utilized is a polynucleotide covalently bonded to a solid resin particle or bead. The second reagent is a labeled restriction enzyme fragment of the normal gene sequence but complementary to a different portion of the target gene, the target binding sites of the two reagents being separated by a restriction enzyme cleavage site. In performing the assay, the sample polynucleotide to be tested is first treated with the specific restriction enzyme and the resulting digest brought into contact with the two reagents under hybridization conditions. Following the hybridization step, the materials bound to the solid phase reagent are separated and washed free of the remaining unbound labeled reagent. The presence of any label bound to the solid support is then determined. While this method affords relatively high sensitivity, the method is complicated by the required wash steps.
Abbott et al. U.S. Pat. No. 4,521,521, issued June 4, 1985, describes a highly sensitive and rapid method for quantitatively assaying analytes in liquid media by directly measuring changes in particle size distribution of reagent particles having analyte insolubilized thereon in a system undergoing antibody-induced aggregation.
There exists a continuing need for a nucleic acid hybridization assay which is highly specific, has enhanced detection sensitivity and is rapid and easy to perform.